The ASC inflammasome adapter governs SAA-derived protein aggregation in inflammatory amyloidosis

Extracellularly released molecular inflammasome assemblies -ASC specks- cross-seed Aβ amyloid in Alzheimer’s disease. Here we show that ASC governs the extent of inflammation-induced amyloid A (AA) amyloidosis, a systemic disease caused by the aggregation and peripheral deposition of the acute-phase reactant serum amyloid A (SAA) in chronic inflammatory conditions. Using super-resolution microscopy, we found that ASC colocalized tightly with SAA in human AA amyloidosis. Recombinant ASC specks accelerated SAA fibril formation and mass spectrometry after limited proteolysis showed that ASC interacts with SAA via its pyrin domain (PYD). In a murine model of inflammatory AA amyloidosis, splenic amyloid load was conspicuously decreased in Pycard−/− mice which lack ASC. Treatment with anti-ASCPYD antibodies decreased amyloid loads in wild-type mice suffering from AA amyloidosis. The prevalence of natural anti-ASC IgG (−logEC50 ≥ 2) in 19,334 hospital patients was <0.01%, suggesting that anti-ASC antibody treatment modalities would not be confounded by natural autoimmunity. These findings expand the role played by ASC and IL-1 independent inflammasome employments to extraneural proteinopathies and suggest that anti-ASC immunotherapy may contribute to resolving such diseases.


Evidence, reproducibility and clarity:
Evidence, reproducibility and clarity (Required) In this manuscript, et al., investigates the role of the inflammasome adapter ASC (in AA amyloidosis).This condition involves the aggregation of serum amyloid A (SAA) and is linked to chronic inflammation.
Firstly, I can directly say that I do recommend this study for publication.This is a well conducted and well-written study which advances the knowledge on IL-1-independent inflammatory functions of inflammasomes.Furthermore, I find it particularly impressive that despite the inflammasome research community is well aware that amyloidosis is a hallmark of inflammatory diseases, it took a neuroscientist specialized in prion diseases to raise the question whether ASC would be involved in seeding serum AA aggregation.

Key findings include:
-ASC forms extracellular aggregates that enhance SAA aggregation, as observed through superresolution microscopy.
-In a mouse model, the absence of ASC significantly reduced amyloid load, not due to increased phagocytosis but likely due to diminished aggregation.
-Treatment with anti-ASC antibodies reduced amyloid load and mitigated weight loss in mice with AA amyloidosis.
These findings suggest that ASC plays a crucial role in AA amyloidosis and that targeting ASC could be a potential therapeutic strategy.The study expands our understanding of the involvement of ASC in proteinopathies beyond neural diseases, pointing to its role in systemic conditions like AA amyloidosis.Main Comments: Overall, the experiments are well-conducted and mostly all controls I would expect were included.With few exceptions, the data is convincing.With that said, I have issues with some of the staining employed in Fig 1 .In Fig. 1, the authors assess ASC staining in cardiac tissues from a patient with vasculitis and systemic inflammation-related AA amyloidosis, and a control patient who died of a heart attack but had no signs of amyloidosis.However, most of the data shown is related to the AL177 anti-ASC.More importantly, no isotype stainings are included.We have previously demonstrated that the AL177 anti-ASC, used here, reacts quite strongly with ASC−/− cells, and it is one of the less specific anti-ASC commercially available (PMID: 27221487).As this is data from one patient (understandably), I wonder if the authors could counterstain ASC in the same samples using a specific human anti-ASC with a different color (ex: Biolegend HASC), and confirm that the signal overlays with the AL-177.
Finally, in Figure 1H it seens from the description that another anti-ASC was used: "referred in the legend as ASC (MAB ASC, Yellow)".Is this a monoclonal anti-ASC?Also, the images show large and bright antibody aggregates (middle of the image, top left corner behind the "H", and a massive fluorescence in the bottom right of the image), indicating the presence of staining artifacts.Again, no counterstaining with isotype controls are shown.
Overall, although I don't dispute the possibility that ASC would co-localize with SAA deposits, I don't think the data presented can safely sustain that claim.I would, therefore, suggest that alternative methods to be employed to substantiate these conclusions: Supposedly, would it be possible to immuno-precipitate (IP) amyloid SAA and assess ASC via western blotting?As well as IP ASC and detect SAA?Or use DSS-crosslinking to find ASC oligomers in tissue areas rich in SAA? **Minor comments:** In addition to these main comments, some minor adjustments are recommended: For example, it would be reasonable to quantify the results in Figure 3G and providing clarification regarding the controls in the figure legend.Though there is significantly less SAA in spleen homogenates from Asc−/−, there also seems to be the case for b-actin in Fig 3G .Moreover, in the figure legend the authors state: "...Spleen homogenate from untreated (-ctrl) and AA+ (+ctrl) C57BL/6 wt mice from an independent experiment served as negative and positive control, respectively."I don't know what the authors mean with that.Is this a montage, or samples from different experiments were run together in one blot?And if so, for what reason?This is confusing and should be clarified.
Furthermore, in the Abstract, a slight rephrasing is suggested to accurately describe ASC specks as molecular aggregates formed inside cells, which are subsequently released into the extracellular space.
Lastly, enhancing the text size in figures, particularly in Fig 3, is advised to improve legibility and overall clarity.

Significance (Required)
In conclusion, this manuscript offers valuable insights into the role of ASC in AA amyloidosis, presenting compelling findings that support its potential as a therapeutic target.Addressing the mentioned concerns and making the suggested revisions will further enhance the manuscript's scientific rigor and impact.Overall, this study is a valuable contribution to the field of inflammasome research and its relevance in systemic conditions like AA amyloidosis.

Estimated time to Complete Revisions (Required) (Decision Recommendation)
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Evidence, reproducibility and clarity (Required)
The manuscript by Losa et al., investigates whether ASC is involved in serum AA amyloidosis.The authors report that ASC colocalizes with SAA in human AA amyloidosis and that purified ASC specks accelerate SAA fibril formation in vitro.In addition, splenic AA amyloid was decreased in Pycard-/-mice compared to Pycard+/+ mice and that treatment with anti-ASC antibodies decreased amyloid loads in Pycard+/+ mice.Lastly, they analyzed serum of 19,334 patients to show that the prevalence of anti-ASC antibodies did not correlate with any specific disease.The authors conclude that ASC to play a role in extraneural proteinopathies of humans and experimental animals and suggest that anti-ASC immunotherapy may contribute to resolving such diseases.The findings in the study are novel and demonstrate a new role for ASC in aggregation proteinopathies.However, there are number of issues that need to be addressed before acceptance for publication.**Major Points:** Figure 3 E depicts Western blots of monomeric SAA in spleen of Pycard+/+ and Pycard-/-mice.The authors should include immunoblots depicting the levels of ASC in these tissues and to demonstrate that the Pycard-/-mice lack ASC.Fig. 3B shows that at 96 hours after injection there was no difference in SAA serum concentration.How do the authors explain this drop in SAA serum concentration?No explanation is provided.
Figure 4 shows anti-ASC administration reduces amyloid load.The immunoblot in Figure 4C does not represent the quantification of the blot.In fact, there are only 3 samples per treatment group whereas the quantification shows 5-6 animals per group.Additionally, the authors have not shown that the drug penetrates the target tissue and how much drug is present in spleen to provide a therapeutic effect.What is the half-life of the drug?These parameters are critical to assess the MOA of the anti-ASC used in these studies.

General Statements
We thank the reviewers for providing valuable comments.We are pleased that our study is considered important to advance the knowledge on IL-1-independent inflammatory functions of inflammasomes.We have clarified and revised the manuscript (track changed) as detailed below in the point-by-point response in this letter.

Referee 1
General: In this manuscript, et al., investigates the role of the inflammasome adapter ASC (in AA amyloidosis).This condition involves the aggregation of serum amyloid A (SAA) and is linked to chronic inflammation.Firstly, I can directly say that I do recommend this study for publication.This is a well conducted and well-written study which advances the knowledge on IL-1-independent inflammatory functions of inflammasomes.Furthermore, I find it particularly impressive that despite the inflammasome research community is well aware that amyloidosis is a hallmark of inflammatory diseases, it took a neuroscientist specialized in prion diseases to raise the question whether ASC would be involved in seeding serum AA aggregation.Key findings include: • ASC forms extracellular aggregates that enhance SAA aggregation, as observed through superresolution microscopy.
• In a mouse model, the absence of ASC significantly reduced amyloid load, not due to increased phagocytosis but likely due to diminished aggregation.
• Treatment with anti-ASC antibodies reduced amyloid load and mitigated weight loss in mice with AA amyloidosis.These findings suggest that ASC plays a crucial role in AA amyloidosis and that targeting ASC could be a potential therapeutic strategy.The study expands our understanding of the involvement of ASC in proteinopathies beyond neural diseases, pointing to its role in systemic conditions like AA amyloidosis.

Significance:
In conclusion, this manuscript offers valuable insights into the role of ASC in AA amyloidosis, presenting compelling findings that support its potential as a therapeutic target.Addressing the mentioned concerns and making the suggested revisions will further enhance the manuscript's

Full Revision
scientific rigor and impact.Overall, this study is a valuable contribution to the field of inflammasome research and its relevance in systemic conditions like AA amyloidosis.
Comment 1: Overall, the experiments are well-conducted and mostly all controls I would expect were included.With few exceptions, the data is convincing.With that said, I have issues with some of the staining employed in Fig 1 .In Fig. 1, the authors assess ASC staining in cardiac tissues from a patient with vasculitis and systemic inflammation-related AA amyloidosis, and a control patient who died of a heart attack but had no signs of amyloidosis.However, most of the data shown is related to the AL177 anti-ASC.More importantly, no isotype stainings are included.We have previously demonstrated that the AL177 anti-ASC, used here, reacts quite strongly with ASC−/− cells, and it is one of the less specific anti-ASC commercially available (PMID: 27221487).As this is data from one patient (understandably), I wonder if the authors could counterstain ASC in the same samples using a specific human anti-ASC with a different color (ex: Biolegend HASC), and confirm that the signal overlays with the AL-177.

Response:
We conducted additional experiments to address the anti-ASC antibody specificity, as now described in Results, Method, and Fig. S1.We tested a set of anti-ASC antibodies (AL177, MY6745, 1C3D7) for their ASC specificity.We confirmed that both the AL177 and the MY6745 antibodies have high ASC-specificity (Fig. S1A).Moreover, for illustration purposes (and to warn other scientists), we included a third anti-ASC antibody (1C3D7) found to be unspecific as it yielded a strong signal in PYCARD -/-(ASC -/-) THP-1 cells (Fig. S1B).In addition, isotype controls were included in these experiments (Fig. S1A, right panels), as suggested by the reviewer, showing no target protein detection in both, PYCARD +/+ (ASC +/+ ) and PYCARD -/-cells underscoring the anti-ASC specificity of AL177 and MY6745 antibodies.
Comment 2: Finally, in Figure 1H it seens from the description that another anti-ASC was used: "referred in the legend as ASC (MAB ASC, Yellow)".Is this a monoclonal anti-ASC?Also, the images show large and bright antibody aggregates (middle of the image, top left corner behind the "H", and a massive fluorescence in the bottom right of the image), indicating the presence of staining artifacts.Again, no counterstaining with isotype controls are shown.

Response:
We apologize for the confusing jargon in Figure 1H."MAB ASC" refers to the anti-ASC PYD antibody (MAB/MY6745).We have corrected the antibody terminology in the legend.MAB/MY6745 is a monoclonal antibody generated by Mabylon that is highly reactive to both human and murine ASC.This antibody was generated to 1) perform an immunotherapy in vivo study and to 2) be used as alternative specific antibody in addition to AL177 to show co-localization of SAA and ASC in a human AA patient using STED superresolution microscopy.MAB/MY6745 is a rabbit monoclonal anti-ASC antibody targeting the pyrin domain (PYD) from which the rabbit Fcγ domain was replaced with that of a mouse IgG2a domain to avoid xenogeneic anti-drug responses in recipients and to improve its effector functions in vivo.To examine possible staining artefacts which can occur with Formalin-Fixed Paraffin-Embedded (FFPE) human tissues, we assessed the specificity of a variety of anti-ASC antibodies (Fig. S1).Our data presented in Fig. S1 show that the monoclonal anti-ASC antibody binds specifically.It is conceivable that AL177 and MAB/MY6745 target different epitopes of ASC, resulting in different staining patterns.An isotype control, included in Fig. S1, was used to test the specificity of the secondary antibodies, and did not show any nonspecific staining.We have adapted and added this to the text body and figure legend accordingly.
Comment 3: Overall, although I don't dispute the possibility that ASC would co-localize with SAA deposits, I don't think the data presented can safely sustain that claim.I would, therefore, suggest that alternative methods to be employed to substantiate these conclusions: Supposedly, would it be possible to immuno-precipitate (IP) amyloid SAA and assess ASC via western blotting?As well as IP ASC and detect SAA?Or use DSS-crosslinking to find ASC oligomers in tissue areas rich in SAA?
Response: In addition to assessing co-localization by means of STED superresolution microscopy (Fig. 1), we also employed LiP-MS with various forms of ASC (monomeric and ASC specks) and identified a previously unrecognized biophysical interaction of SAA and the ASC PYD domain (Fig. 2C-F).As an orthogonal line of evidence, we provided kinetic data showing that SAA aggregation is enhanced in the presence of ASC specks (Fig. 2A-B).We feel that these results are reasonably convincing, but we agree that co-localization is almost invariably an aspirational finding, and even superresolution microscopy cannot fully exclude the presence artifacts (nor can, in fairness, co-immunoprecipitation, which must often rely on overexpression).A sentence acknowledging this limitation was added to the Discussion.Comment 4: For example, it would be reasonable to quantify the results in Figure 3G and providing clarification regarding the controls in the figure legend.Though there is significantly less SAA in spleen homogenates from Asc−/−, there also seems to be the case for b-actin in Fig 3G .Moreover, in the figure legend the authors state: "...Spleen homogenate from untreated (-ctrl) and AA+ (+ctrl) C57BL/6 wt mice from an independent experiment served as negative and positive control, respectively."I don't know what the authors mean with that.Is this a montage, or samples from different experiments were run together in one blot?And if so, for what reason?This is confusing and should be clarified.

Response:
We reworded the figure legend to provide clarity about the technical assay controls and adjusted the labels in Fig. 3E accordingly: To ascertain SAA antibody functionality, mouse spleen homogenate from independently obtained and Congo red-confirmed AA + tissue served as positive, whereas non-induced (AA -) spleen tissue served as negative technical controls.(Fig 3E).We decided to show the two (positive/AA + and negative/AA -) technical controls in Fig. 3E.
Comment 5: Furthermore, in the Abstract, a slight rephrasing is suggested to accurately describe ASC specks as molecular aggregates formed inside cells, which are subsequently released into the extracellular space.
Response: We thank the referee for bringing this to our attention.We rephrased the abstract accordingly.

Referee 2
General: The manuscript by Losa et al., investigates whether ASC is involved in serum AA amyloidosis.The authors report that ASC colocalizes with SAA in human AA amyloidosis and that purified ASC specks accelerate SAA fibril formation in vitro.In addition, splenic AA amyloid was decreased in Pycard-/-mice compared to Pycard+/+ mice and that treatment with anti-ASC antibodies decreased amyloid loads in Pycard+/+ mice.Lastly, they analyzed serum of 19,334 patients to show that the prevalence of anti-ASC antibodies did not correlate with any specific disease.The authors conclude that ASC to play a role in extraneural proteinopathies of humans and experimental animals and suggest that anti-ASC immunotherapy may contribute to resolving such diseases.The findings in the study are novel and demonstrate a new role for ASC in aggregation proteinopathies.However, there are number of issues that need to be addressed before acceptance for publication.

Significance:
The findings in the study are novel and demonstrate a new role for ASC in aggregation proteinopathies.This study reports a crucial role for ASC in SAA interaction and recruitment, SAA serum level modulation, SAA fibril formation acceleration, and controlling the extent of inflammation associated amyloidosis with respect to AA amyloid deposition Comment 1: Figure 3 E depicts Western blots of monomeric SAA in spleen of Pycard+/+ and Pycard-/mice.The authors should include immunoblots depicting the levels of ASC in these tissues and to demonstrate that the Pycard-/-mice lack ASC.

Response:
We did not perform ASC immunoblots for Pycard -/-and Pycard +/+ mice since the absence of the ASC protein in this well-established mouse line has been demonstrated in several key publications, including under inflammation conditions (right side of the figure below, from Mariathasan et al., Nature, 2014).However, we show ASC IHC of Pycard +/+ and Pycard -/-AA + mice on spleen, confirming the absence of an ASC signal in Pycard -/-mice and its presence in the Pycard +/+ (Fig. 3F).Moreover, our genotyping data confirmed the presence and absence of the Pycard gene in Pycard +/+ and Pycard -/-AA + mice.
Response: To assess the pharmacokinetics of the anti-ASC antibody, we determined its titers in serum by ELISA at various time points up to 96 hpi after the first injection.The anti-ASC antibody serum levels peaked at 24 hpi and declined to about half maximal serum concentration levels at 96 hpi.This serum half-life, for the injected concentration, is in the range of reported kinetic parameters of engineered monoclonal antibodies (e.g., Unverdorben et al., MAbs, 2016;Foss et al., Nat Comm, 2024) (Fig. 4B).Because of the high permeability of splenic red pulp vasculatures, and because of the absence of any selectively permeable barrier, efficacious imbibement of the splenic extracellular space can be plausibly expected.Theoretically, one could perfuse mice intracardially with PBS and then measure antibody in tissue.Such measurements can work relatively well in the brain, which possesses a highly impermeable barrier.However, here we would find it difficult to convince ourselves that such measurements would not be contaminated by residual blood in splenic capillaries that may be difficult to clean up through perfusion.Therefore, we did not measure the antibody levels in the spleen.

Full Revision
Comment 6: Methods: The authors provide the number of animals employed in the Supplemental Tables 5 and 7.These numbers should be provided in the methods section or in the Figure legends.Additionally, how many replicates were performed for the data in Figure 2?
Response: As suggested by the reviewer we now provide the number of animals in the figure legends of main Fig. 2 and Fig. 3 in addition to those in Table 5 and Supp Table 7 to enhance clarity.

Referee 3
General: The manuscript by Losa et al. explores the co-aggregation of ASC with serum amyloid A (SAA) in vivo and in mouse models, It posits that, similar to Amyloid beta, SAA is cross-seeded by ASC foci both in vitro and in vivo.This review only addresses the co-localization and in vitro cross seeding data (Figs. 1  and 2A, B), not the mouse experiments or mass spectrometry data.The manuscript first shows co-deposition of ASC with SAA amyloid.SAA was stained both with Congo red and ThS, both standard dyes for amyloid staining.Figure S2 shows CR birefringence, the hallmark of amyloid deposits.The authors then move to demonstrate co-localization of SAA and ASC in confocal and STED immuno-fluorescence microscopy.

Significance:
The discovery of the role of ASC in Alzheimer's disease generated an exciting new hypothesis to the etiology of sporadic AD, for which the cause is unknown.The current manuscript finds that ASC may also play a role in AA amyloidosis, which is a significant finding.
Comment 1: Confocal images C-E show overlapping staining of markers for both SAA and ASC.Similarly, STED images show co-aggregation of ASC and SAA in amyloidosis patients.However, since confocal images F and G seem to show overlapping staining of the yellow and magenta channels as well, a careful quantitative analysis of the data I needed.Quantify co-localization (Pearson coefficient) in confocal and STED images.STED images from control patients are missing and need to be included.
Response: AA amyloidosis is a relatively rare disease, and tissue samples thereof are even rarer.We only had access to the samples of one patient in both control and SAA groups.This limitation prevented us from conducting quantitative analyses.Rather than looking at the Pearson -or, possibly better, Spearman -correlation coefficient, we opted for an unbiased method of correlation in which we reconstructed the picture using 3D surface rendering with the Imaris software (see Fig. 1).From this reconstruction, we exported the barycenter of each surface on a 3D plot for both SAA and ASC markers (see Fig. S2B-C).Each point represents the center of a surface, while the box plots on the sides represent the distribution of the markers in space, demonstrating the overlap of the markers for ASC and SAA.We also understand the suggestion to conduct STED imaging on control samples to show the absence of coaggregation.However, we could not be sure of which region to capture and how to decide on the focus, as we did not detect strong signal from confocal images of the control sample.Imaging blindly would almost necessarily lead to irrelevant imaging and aberrant comparison.We do not claim any quantitative data out of these images; however, we report an observation.Quantitative and mechanistic co-aggregation data are presented in Fig. 2 using LiP-MS.

Comment 2:
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In which section is the information available?
(Reagents and Tools

Plants and microbes
Information included in the manuscript?
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(Reagents and Tools

Core facilities Information included in the manuscript?
In which section is the information available?
(Reagents and Tools If your work benefited from core facilities, was their service mentioned in the acknowledgments section?Yes In acknowledgments section.

Design
-common tests, such as t-test (please specify whether paired vs. unpaired), simple χ2 tests, Wilcoxon and Mann-Whitney tests, can be unambiguously identified by name only, but more complex techniques should be described in the methods section; Please complete ALL of the questions below.Select "Not Applicable" only when the requested information is not relevant for your study.
if n<5, the individual data points from each experiment should be plotted.Any statistical test employed should be justified.Source Data should be included to report the data underlying figures according to the guidelines set out in the authorship guidelines on Data Each figure caption should contain the following information, for each panel where they are relevant: a specification of the experimental system investigated (eg cell line, species name).the assay(s) and method(s) used to carry out the reported observations and measurements.an explicit mention of the biological and chemical entity(ies) that are being measured.an explicit mention of the biological and chemical entity(ies) that are altered/varied/perturbed in a controlled manner.Studies involving human participants: State details of authority granting ethics approval (IRB or equivalent committee(s), provide reference number for approval.

Yes
Material and methods section.
Studies involving human participants: Include a statement confirming that informed consent was obtained from all subjects and that the experiments conformed to the principles set out in the WMA Declaration of Helsinki and the Department of Health and Human Services Belmont Report.

Yes
Material and methods section.
Studies involving human participants: For publication of patient photos, include a statement confirming that consent to publish was obtained.

Yes
Material and methods section.
Studies involving experimental animals: State details of authority granting ethics approval (IRB or equivalent committee(s), provide reference number for approval.Include a statement of compliance with ethical regulations.

Yes
Material and methods section.
Studies involving specimen and field samples: State if relevant permits obtained, provide details of authority approving study; if none were required, explain why.

Not Applicable
Dual Use Research of Concern (DURC) Information included in the manuscript?
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(Reagents and Tools

Reporting
Adherence to community standards Information included in the manuscript?
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(Reagents and Tools Have primary datasets been deposited according to the journal's guidelines (see 'Data Deposition' section) and the respective accession numbers provided in the Data Availability Section?

Yes
Described in material and methods section.
Were human clinical and genomic datasets deposited in a public accesscontrolled repository in accordance to ethical obligations to the patients and to the applicable consent agreement?

Not Applicable
Are computational models that are central and integral to a study available without restrictions in a machine-readable form?Were the relevant accession numbers or links provided?

Yes
Described in material and methods section.
If publicly available data were reused, provide the respective data citations in the reference list.

Not Applicable
The MDAR framework recommends adoption of discipline-specific guidelines, established and endorsed through community initiatives.Journals have their own policy about requiring specific guidelines and recommendations to complement MDAR.

Comment 6 :
Lastly, enhancing the text size in figures, particularly in Fig 3, is advised to improve legibility and overall clarity.Response:The legibility and style of main Fig.3text sizes has been changed and additional figure formatting has been performed.
Figures are not edited by the production team.All lettering should be the same size and style; figure panels should be indicated by capital letters (A, B, C etc).Gridlines are not allowed except for log plots.Figures should be numbered in the order of their appearance in the text with Arabic numerals.Each Figure must have a separate legend and a caption is needed for each panel.*Additionalimportant information regarding figures and illustrations can be found at Table, Materials and Methods, Figures, Data Availability Section) Table, Materials and Methods, Figures, Data Availability Section) Table, Materials and Methods, Figures, Data Availability Section)

In which section is the information available?
Table, Materials and Methods, Figures, Data Availability Section) (Reagents and Tools Table, Materials and Methods, Figures, Data Availability Section) Provide species, strain, sex, age, genetic modification status.Provide accession number in repository OR supplier name, catalog number, clone number, OR RRID.

In which section is the information available?
Table, Materials and Methods, Figures, Data Availability Section) (Reagents and Tools Table, Materials and Methods, Figures, Data Availability Section) If collected and within the bounds of privacy constraints report on age, sex and gender or ethnicity for all study participants.
Table, Materials and Methods, Figures, Data Availability Section)

Checklist for Life Science Articles (updated January Study protocol Information included in the manuscript? In which section is the information available?
ideally, figure panels should include only measurements that are directly comparable to each other and obtained with the same assay.plotsincludeclearly labeled error bars for independent experiments and sample sizes.Unless justified, error bars should not be shown for technical the exact sample size (n) for each experimental group/condition, given as a number, not a range; a description of the sample collection allowing the reader to understand whether the samples represent technical or biological replicates (including how many animals, litters, cultures, etc.).a statement of how many times the experiment shown was independently replicated in the laboratory.This checklist is adapted from Materials Design Analysis Reporting (MDAR) Checklist for Authors.MDAR establishes a minimum set of requirements in transparent reporting in the life sciences (see Statement of Task: 10.31222/osf.io/9sm4x).Please follow the journal's guidelines in preparing your the data were obtained and processed according to the field's best practice and are presented to reflect the results of the experiments in an accurate and unbiased manner.(ReagentsandTools Table, Materials and Methods, Figures, Data Availability Section)If study protocol has been pre-registered, provide DOI in the manuscript.For clinical trials, provide the trial registration number OR cite DOI.

In which section is the information available?
(Reagents and Tools Table, Materials and Methods, Figures, Data Availability Section)

Sample definition and in-laboratory replication Information included in the manuscript? In which section is the information available?
(Reagents and Tools Table, Materials and Methods, Figures, Data Availability Section)

In which section is the information available?
(Reagents and Tools Table, Materials and Methods, Figures, Data Availability Section)

granting approval and reference number for
Table, Materials and Methods, Figures, Data Availability Section) Could your study fall under dual use research restrictions?Please check biosecurity documents and list of select agents and toxins (CDC): https://www.selectagents.gov/sat/list.htmNot Applicable If you used a select agent, is the security level of the lab appropriate and reported in the manuscript?Not Applicable If a study is subject to dual use research of concern regulations, is the name of the authority the regulatory approval provided in the manuscript?

and III randomized controlled trials
Table, Materials and Methods, Figures, Data Availability Section) State if relevant guidelines or checklists (e.g., ICMJE, MIBBI, ARRIVE, PRISMA) have been followed or provided.Not Applicable For tumor marker prognostic studies, we recommend that you follow the REMARK reporting guidelines (see link list at top right).See author guidelines, under 'Reporting Guidelines'.Please confirm you have followed these guidelines., please refer to the CONSORT flow diagram (see link list at top right) and submit the CONSORT checklist (see link list at top right) with your submission.See author guidelines, under 'Reporting Guidelines'.Please confirm you have submitted this list.Reagents and Tools Table, Materials and Methods, Figures, Data Availability Section) (